Research supported by this grant over the past two decades, has focused on using the oncogenic protein tyrosine kinase, Src, as a molecular guide to identify tyrosine phosphorylated proteins that play key roles in cellular signaling. Characterization of so-called "Src targets" has led to the identification of a number of cellular proteins, such as Focal Adhesion Kinase (FAK), p130Cas and paxillin, proteins that function within the context of specific actin-based cytoskeletal structures, e.g., focal adhesions, as well as Cortactin, a protein that localizes predominantly to cortical actin, a site of dynamic actin remodeling. The long range goal of the studies outlined in this proposal is to understand how proteins such as Cortactin, function in the context of the cortical actin cytoskeleton to integrate cell surface receptor mediated cell signaling in a "temporal and spacial" context. Information garnered over the past granting period shows that the Src-substrate Cortactin is a multifunctional adapter protein that interacts on the one hand with the "cortical actin network" present in filopodia and lamellipodia by binding the Arp2/3 complex and links via its carboxyl-terminal SH3 domain to a number of functionally interesting and potentially important signaling and scaffolding proteins. This revised application again outlines four specific aims to address the function of Cortactin: In Aim 1 we will use molecular genetic, cellular and biochemical approaches to study the factors that regulate the interaction of Cortactin with the Arp2/3 complex of proteins. In Aim 2, we will extend and further develop our studies aimed at identifying and characterizing proteins that interact with the Cortactin SH3 domain. We will continue to characterize proteins that function as binding partners for Cortactin, including two recently identified SH3 effectors, WIP, Wasp Interacting Protein, a regulator of Arp2/3-dependent actin polymerization and dynamin, a GTPase implicated in the regulation of endocytic and secretory pathways. We will also extend and further develop studies to identify possible interactions of cellular proteins with the "helical-proline-ser-rich" region of Cortactin, here-to-fore unstudied interaction domain. In Aim 3 we will examine how tyrosine phosphorylation influences the interactions of Cortactin with known cellular binding partners. Finally in Aim 4, we will examine whether expression of full length Cortactin or predicted dominant inhibitory forms of Cortactin function to alter the dynamics of EGF-induced lamellipodia formation or cell migration. We will also determine whether Cortactin or Cortactin variants influence the dynamics of growth factor receptor signaling to downstream effectors, with particular attention to Ras signaling and EGF receptor internalization. Finally we propose to generate mice bearing a genetically altered allele of Cortactin, allowing for Cre-mediated excision and replacement of the Cortactin SH3 domain. Such mice, bearing a mutated form of Cortactin will allow one to directly test the role of Cortactin in mediating specific cellular functions in a cell type specific fashion.